PROJECTSUMMARY Drugdiscoverypipelinesforneuropsychiatricdisordersaredry.Oneapproachtorejuvenatingthese pipelineswouldbetocreateassaysbasedonrelevantdiseasephenotypesinprimaryneurons,somethingthat iscurrentlylacking.However,ascalableassaydevelopmentplatformthatisbasedonbonafideneurons, remainscosteffective,andthatcansupportindustriallevelhigh-throughputscreening(HTS)doesnotcurrently exist.Overthepasteightyears(spreadacrossdifferentNIH-sponsoredgrants),ourcollaborativegrouphas createdaflexibleandscalableprimaryneuron-basedassaydevelopmentsystemthatiscompatiblewith industrial-levelHTS.Ourlong-standinggoalforthisprojecthasbeentooptimizetheseproceduresand workflowstosupportneuron-basedHTSphenotypicassayssothattheycansupportverylarge screeningcampaignsofupto200Kcompounds. Wearehappytoreportthatprogressoverthelastbudgetperiodhaspushedusclosertowardthis statedgoal.Wehaveinventedastate-of-the-art,disease-modelingassaycreatedinprimaryneuronsthatis designedtodiscovercompoundsthatreversethecellularconsequencesofgenetichaploinsufficiency.Indeed, asubstantialproportionofchildhoodbraindisordersarecausedbysingleautosomaldominantvariants resultingingenetichaploinsufficiency.Theraregeneticbraindisordersthatarisefromthesevariantsoffer greatpotentialfortranslationbecausethediseasemechanismiswell-understood(i.e.lowproteinexpression). Therefore,arationaleprecisiontherapyfortreatinggenetichaploinsufficiencydisorderswouldbetodiscover ?magicbullet?compoundsthatraiseexpressionoffunctionalproteinsfromtheremainingundamagedallele (e.g.?boostingcompounds?).Inthisrenewalproject,wewillemploytechnicalinnovationsthathaveunlocked thescalabilityofprimaryneuronsforphenotypicHTS.Asaproof-of-principle,wewillscale-upand implementanassaythatreportsreversaloflowSynGAPexpressioninneuronscausedbygenetic haploinsufficiencyoftheSYNGAP1/Syngap1gene.WewillminiaturizeanHTS-compatibleanddisease- modelingsteady-stateendogenousSynGAPexpressionassaysothatitiscompatiblewithindustrialscaleHTS automation.Onceimplemented,wewillthenscreenupto200,000uniquesubstancesusingacompletely automatedversionoftheneuron-basedSynGAPexpressionassay.Finally,usingacomprehensivemulti-stage biologicalvalidationfunnel,wewillidentifyandprioritizethemosttranslatablechemicalprobesthatraise SynGAPproteinexpression.Theoverallimpactofthisprojectisthatdiscoveryofmultiple,validatedSynGAP boostingcompoundswouldprovideproof-of-principlethatourflexibleplatformisaneffectivetoolfor phenotypicdrugdiscoveryfornervoussystemdisorders.